Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma

The Plasmodium falciparum circumsporozoite (CS) protein-based pre-erythrocytic stage vaccine, RTS,S, induces a high level of protection against experimental sporozoite challenge. The immune mechanisms that constitute protection are only partially understood, but are presumed to rely on Abs and T cell responses. In the present study we compared CS protein peptide-recalled IFN-gamma reactivity of pre- and RTS,S-immune lymphocytes from 20 subjects vaccinated with RTS,S. We observed elevated IFN-gamma in subjects protected by RTS,S; moreover, both CD4(+) and CD8(+) T cells produced IFN-gamma in response to CS protein peptides. Significantly, protracted protection, albeit observed only in two of seven subjects, was associated with sustained IFN-gamma response. This is the first study demonstrating correlation in a controlled Plasmodia sporozoite challenge study between protection induced by a recombinant malaria vaccine and Ag-specific T cell responses. Field-based malaria vaccine studies are in progress to validate the establishment of this cellular response as a possible in vitro correlate of protective immunity to exo-erythrocytic stage malaria vaccines.     

Changes in germ cell population in young and adult female mice from two inbred strains: CBA/Kw and KE

Female mice from two inbred strains CBA/Kw and KE differ markedly in fertility. The gametes of females from KE strain are of poorer quality than those of CBA/Kw. We analyzed the number of oocytes per ovary in KE and CBA/Kw mice aged 5, 25, 90, 180 and 360 days. The ovaries were dissected and processed according to the routine histological methods. In case of five-day-old females we used a modified distributed point counting method while in order to examine the gonads of older females, the nucleoli counting method was applied. In general, we observed gradual decrease in germ cell number throughout the whole life of females from both strains. The noticeable wave of oocyte loss occurs between 5th and 25th days of life. The mice from KE inbred strain on day 25th (1650 +/- 322 vs. 1140 +/- 210) and 90(th) (1040 +/- 211 vs. 692 +/- 89) days have significantly (p<0.005) more germ cells than the females from CBA/Kw strain. In older females the differences were not statistically significant. Interestingly, CBA/Kw females were found to have more rapid loss of primordial follicles throughout their lives. This can explain their shortened reproductive lifespan which was observed earlier.     

A competitor DNA template for the molecular quantification of the hepatitis B virus

The molecular determination of viral load in the serum represents the most valuable prognostic marker of HBV infection. In this paper, a new molecular assay for the quantitative measurement of HBV presence is described. It is based on PCR performing with a HBV-specific competitor DNA template. For the construction of the DNA template, a HBV DNA-originated 436 bp DNA fragment was modified by introducing a 110 bp deletion and cloned into pUC19. The resulting vector serves as the competitor DNA template in the competitive PCR. Post-PCR, the competitor DNA generates an amplified fragment of 306 bp; it could be easily distinguished from the product generated from the viral-originated DNA product (416 bp) when the same primers are used. The quantitative ratio between the two products enables the quantitative determination of viral load. The range of the HB-PCR assay is from 3 x 10(4)to 6 x 10(10) particles/ml. A serum HBV load determination performed by HB-PCR assay indicated a close correlation with the results of the Quantiplex HBV DNA assay (bDNA). The HB-PCR assay is cheap, reliable and easy to use in any laboratory working with PCR methods.     

DNase I and II present in avian oocytes: a possible involvement in sperm degradation at polyspermic fertilisation

During polyspermic fertilisation in birds numerous spermatozoa enter the eggs, in contrast to the situation in mammals where fertilisation is monospermic. However, in birds only one of the spermatozoa which have entered an egg participates in zygote nucleus formation, while the supernumerary spermatozoa degenerate at early embryogenesis. Our previous work has demonstrated the presence in preovulatory quail oocytes of DNase I and II activities able to digest naked lambdaDNA/HindIII substrate in vitro. In the present studies, the activities of both DNases in quail oocytes at different stages of oogenesis and in ovulated mouse oocytes were assayed in vitro using the same substrate. Degradation of quail spermatozoa by quail oocyte extracts was also checked. Digestion of the DNA substrate was evaluated by electrophoresis on agarose gels. The activities of DNase I and II in quail oocytes increased during oogenesis and were the highest in mature oocytes. The activities were present not only in germinal discs but also in a thin layer of cytoplasm adhering to the perivitelline layer surrounding the yolk. At all stages of oogenesis the activity of DNase II was much higher than that of DNase I. DNA contained in spermatozoa was also degraded by the quail oocyte extracts under conditions optimal for both DNases. In contrast to what is observed in quail oocytes, no DNase activities were detected in ovulated mouse eggs; this is logical as they would be useless or even harmful in monospermic fertilisation. The possible role of DNase activities in avian oocytes, in degradation of accessory spermatozoa during polyspermic fertilisation, is discussed.     

Three novel mutations in exon 21 encoding beta-cardiac myosin heavy chain

Familial hypertrophic cardiomyopathy has a complex multigenic background. Previous work allowed to determine one of the gene loci responsible for this disease on chromosome 14 band q11-q12, and linked it to the alpha and beta-cardiac myosin heavy chains. In this study we demonstrate changes in exon 21, coding for beta-myosin heavy chain. We described 4 patients from different families with an unequivocal diagnosis of hypertrophic cardiomyopathy based on the clinical picture. Direct sequencing of exon 21 revealed the presence of 5 novel mutations. Two of the mutations in codons 771 and 781 revealed in our study did not result in any changes in amino acid sequence. The next three were as follows: in codon 782 (AGC > GAC) transition responsible for Ser-->Asp substitution; in codon 779 (GAG > TAG) mutation that results in replacement of Glu-->Stop; in codon 774 (GAG > GTG) which is expressed as substitution of Glu-->Val. These mutations are located close to mutations identified and described in the literature, so they are likely to cause similar symptoms.     

Ecological attributes recorded in stable isotope ratios of arboreal prosimian hair

Carbon and nitrogen stable isotope ratios were measured in hair samples from two species of Galago from Gedi Ruins National Monument in eastern Kenya and from Lepilemur leucopus from Beza Mahafaly Special Reserve in southern Madagascar. Forest structure was generally similar in the two areas but average rainfall was lower in Madagascar. Species average ¹³C values varied with feeding height in the forest canopy and with average rainfall level as expected from reported variation in plant ¹³C values. G. garnettii, which feeds higher in the forest canopy, had less negative ¹³C values than G. zanzibaricus, which spends more time below 5 m. L. leucopus, from a drought-afflicted forest, had less negative hair ¹³C values than the two galago species. The values within the Lepilemur sample showed a positive linear relation with percent dependence on a CAM tree species and with xeric conditions within the species reserve. Nitrogen stable isotope ratios varied with trophic level of feeding and with time spent feeding on leguminous plants. The insectivorous galagos had significantly more positive ¹⁵N values than the folivorous L. leucopus. Within the Lepilemur sample, ¹⁵N values varied inversely with the percent of feeding time spent on leguminous plants. The range of ¹⁵N and ¹³C values in each of the prosimian species is larger than reported for animals fed monotonous diets and for New World monkey species. The monkey species feed as groups of individuals whereas the prosimians have solitary feeding habits. The ranges in the prosimian species apparently reflect the greater variation in diet among individual prosimians compared to individual monkeys. The isotope data reported here are equivalent, on average, to those reported for other arboreal species from similar forest habitats and with similar dietary habits. This supports the use of such data for paleoecological reconstruction of forest and woodland systems and diet reconstruction of extinct primate populations and species. Received: 18 April 1997 / Accepted: 11 August 1997     

Role of glucose in interleukin-1β production by lipopolysaccharide-activated human monocytes

When monocytes are activated with endotoxin (lipopolysaccharide [LPS]), they make and release several mediators, including interleukin-1β (IL-1β). This study was undertaken to investigate the role of glucose in IL-1β production by these cells. IL-1β was produced in a dose-dependent manner to glucose concentration in the culture medium. The uptake of (³H)2-deoxyglucose in monocytes was stimulated by LPS 1,554% after 10 minutes, 6,095% after 2 hours, then gradually declined after 4 hours of incubation. The inhibition of the uptake of (³H)2-deoxyglucose by either 10 μM cytochaiasin B or phloretin, added at the time of monocyte activation, was accompanied by significant reduction in ATP/ADP ratio and the inhibition of the production of IL-1β by activated monocytes. The synthesis of total protein did not change in monocytes activated in the absence of glucose in the culture medium, nor in the presence of either 10 μM cytochalasin B or phloretin. The export of IL-1β from LPS-activated monocytes was not inhibited by either 10 μM cytochalasin B or phloretin, nor in the absence of glucose in the culture medium. These data suggest that (1) glucose is required for LPS-induced IL-1β production by monocytes; (2) glucose is the major source of ATP for IL-1β production; (3) glucose transporter (GLUT 1) does not control the export of IL-1β. © 1993 Wiley-Liss, Inc.     

Enantioselective synthesis and cytotoxic evaluation of 4,5-dihydro-5-[aryl(hydroxy)methyl]-3-methylidenefuran-2(3H)-ones

A series of enantiomerically enriched 4,5-dihydro-5-[aryl(hydroxy)methyl]-3-methylidenefuran-2(3H)-ones (8) were synthesized by means of asymmetric Sharpless dihydroxylation of the 2-phosphorylated 5-aryl-pent-4-enoic acids 13, followed by Horner-Wadsworth-Emmons reaction of the resulting furanones 15 (Scheme 2). An enantiomeric excess (ee) of 20-95% was achieved for compounds 8, and their absolute configurations were determined by the Mosher ester method. Cytotoxic evaluation against L-1210 and HL-60 leukemia cell lines revealed that the target compounds 8 are active in the micromolar concentration range (Table 2). Thereby, significant differences in activity between the corresponding enantiomers were observed for the HL-60 cell line.     

The structure of Yersinia pestis V-antigen, an essential virulence factor and mediator of immunity against plague

The LcrV protein (V-antigen) is a multifunctional virulence factor in Yersinia pestis, the causative agent of plague. LcrV regulates the translocation of cytotoxic effector proteins from the bacterium into the cytosol of mammalian cells via a type III secretion system, possesses antihost activities of its own, and is also an active and passive mediator of resistance to disease. Although a crystal structure of this protein has been actively sought for better understanding of its role in pathogenesis, the wild-type LcrV was found to be recalcitrant to crystallization. We employed a surface entropy reduction mutagenesis strategy to obtain crystals of LcrV that diffract to 2.2 A and determined its structure. The refined model reveals a dumbbell-like molecule with a novel fold that includes an unexpected coiled-coil motif, and provides a detailed three-dimensional roadmap for exploring structure-function relationships in this essential virulence determinant.     

Augmentation of hypoxic respiration after brief hyperoxia in the anesthetized cat: Putative function of GABA<Subscript>A</Subscript> neurotransmission

In this study, we attempted to determine the role of GABA neurotransmission in augmentation of hypoxic respiration by antecedent hyperoxic breathing. The experiments were performed in anesthetized, paralyzed and vagotomized cats divided into control and bicuculline (a GABA_A receptor blocker)-injected groups. The experimental protocol consisted of exposing the animals to successive hypoxic-hyperoxic-hypoxic conditions. Respiration was assessed using phrenic electroneurograms, from which the peak phrenic height, a surrogate of the tidal volume component, and respiratory rate were obtained, and their product, the respiratory minute output, was calculated. We found that prior hyperoxic ventilation increased the subsequent respiratory response to hypoxia by an average of 23.5%, compared with the preoxygen response. This increase was driven by volume respiration. The biphasic character of the hypoxic respiratory response, consisting of stimulatory and depressant phases, was sustained. Bicuculline abolished the augmentative effect on hypoxic respiration of prior hyperoxia, which suggests that oxygenation induces GABA_A-mediated hyperexcitability of respiratory neurons, possibly by the liberation of reactive oxygen species. We concluded that GABA neurotransmission is pertinent to the effect of hyperoxia on hypoxic respiratory reactivity.